Protocol for manufacture of RDG conjugated Magnetite cationic Liposomes

1
Protocol for manufacture of RDG conjugated
Magnetite cationic Liposomes

• Andrew Naber
• Dan Knipmeyer

2
Objective

• Develop protocol for enveloping magnetite
  nano-particles with liposomes bound to an RGD
  peptide sequence for cell specific integrin
  targeting.

3
Concepts

• RGD peptides are protein sequences that are
  targeted to specific integrins. By targeting
  endothelial cells we hope to create a
  cell-magnetite complex that can bind to magnetic
  surfaces.

4
Suggested Materials

• Chloroform
• Bath Sonicator
• Vortexor
• Gas tight syringe
• Lipid Mix
• N-(a-trimethylammonioacetyl)-didodecyl-D-glutamate
  chloride (TMAG)
• dilauroylphosphatidyl-choline (DLPC)
• dioleoylphosphatidylethanolamine-N-3-(2-pyridyldi
  thio)-propionate (PDP-DOPE)
• Lipid marker DiI
• 2-morpholinoethanesulfonic acid (MES) buffer (pH
  5.0)
• 10 nm magnetite nano-particles
• Specific RDG peptides
• Round bottom flask

5
Procedure

• Dissolve the three lipids (in a 122 molar
  ratio) into chloroform in the round bottom flask.
  Do this at 10-20 lipid/ml organic solvent.
• Remove solvent either through dry nitrogen method
  (volumelt1ml) or through rotary evaporation.
• Allow the chloroform to evaporate in a vacuum
  (overnight). Lipid film should coat the bottom of
  the flask.
• Suspend the magnetite nano-particles in water.
• Hydrate dried lipid layer on the bottom of the
  flask with nano-particle suspended water.

6
Procedure cont.

• Vortex the lipid/water/nano-particle solution for
  a while.
• Sonicate solution for 30 mins at 28 W in a bath
  sonicator to reduce to single laminar liposomes.
• Add 2ml of purified MCLs to the RGD peptides in a
  .55 peptide to 1 PDP-DOPE molar ratio.
• Gently agitate this solution for 3.5 hours in a
  solution of 2-morpholinoethanesulfonic acid (MES)
  buffer (pH 5.0).

7
Illustration
8
Purify the MCLs

• At this point there will be a mixture of
  liposomes that contain magnetite, liposomes
  without magnetite, and magnetite nano-particles.
• Place mixture in a magnetic stand and vacuum out
  particles that do not stick to the sides.
• The remaining nano-particles in suspension should
  precipitate after a while.

9
Next Phase

• Cell Adhesion Test
• Adhesion Test in flow chamber